title: BMP7 gene transfer via gold nanoparticles into stroma inhibits corneal fibrosis in vivo.
abstract: This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7's anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10(4) gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.
authors.affiliation: Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America.
authors.forename: Ashish
authors.initials: A
authors.lastname: Tandon
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.forename: Jason T
authors.initials: JT
authors.lastname: Rodier
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Klibanov
authors.forename: Frank G
authors.initials: FG
authors.lastname: Rieger
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D003315:Cornea; D003316:Corneal Diseases / Q000473:pathology / Q000628:therapy*; D003319:Corneal Stroma / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005355:Fibrosis; D018628:Gene Dosage; D015316:Genetic Therapy; D006046:Gold; D006801:Humans; D053768:Metal Nanoparticles; D011817:Rabbits; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D014162:Transfection; D016896:Treatment Outcome
chemical_list: D055419:Bone Morphogenetic Protein 7; D051785:Smad Proteins; D006046:Gold